Ferroptosis in Arthritis: Driver of the Disease or Therapeutic Option?

Ferroptosis is a form of iron-dependent regulated cell death caused by the accumulation of lipid peroxides. In this review, we summarize research on the impact of ferroptosis on disease models and isolated cells in various types of arthritis. While most studies have focused on rheumatoid arthritis (RA) and osteoarthritis (OA), there is limited research on spondylarthritis and crystal arthropathies. The effects of inducing or inhibiting ferroptosis on the disease strongly depend on the studied cell type. In the search for new therapeutic targets, inhibiting ferroptosis in chondrocytes might have promising effects for any type of arthritis. On the other hand, ferroptosis induction may also lead to a desired decrease of synovial fibroblasts in RA. Thus, ferroptosis research must consider the cell-type-specific effects on arthritis. Further investigation is needed to clarify these complexities.


Introduction
Ferroptosis is an iron-dependent form of regulated cell death characterized by lipid hydroperoxide accumulation.The term was coined in 2012 by Stockwell et al. to describe a form of cell death induced by an inhibition of cysteine manifesting an abnormal mitochondrial structure [1,2].Although extensively studied in cancer cells as a potential therapeutic target [3], its role in autoimmune and autoinflammatory diseases involving arthritis remains less clear despite the fact that iron accumulation in inflamed tissue leads to cellular toxicity.In line with this finding, iron overload has also been found to play a role in the arthritic joint [4].Further, since the joint remains a hypoxic environment despite the fact that hypoxia-inducible factors induce angiogenesis to some extent, oxidative stress may play an important role in the synovial tissue [5].Thus, the question arose, whether iron excess and oxidative stress might lead to ferroptosis in the inflamed joint.Alternatively, iron chelation is experimentally used as a mimic of hypoxia response and could serve as an alternative explanation for several findings in arthritis [6].In recent years, the relationship between inflammation and ferroptosis has been explored in several ways for a better understanding of diverse pathomechanisms in inflammatory diseases, since ferroptosis is accompanied by the release of pro-inflammatory molecules, such as interleukin (IL)-1β and IL-18 [7].Thus, research on ferroptosis in diseases involving arthritis has taken its path in order to find new therapeutic targets for these in part still not adequately treatable diseases.
This review aims to elucidate the role of ferroptosis in arthritis and its therapeutic implications.For the search conducted in this review, we used the title and abstract key terms ""ferroptosis" and "arthritis" or "rheumatoid arthritis" or "osteoarthritis" or "spondylarthritis" or "cristal arthropathies" or "gout"" in PubMed.

Regulating Ferroptosis
Ferroptosis is distinct from other forms of regulated cell death like apoptosis, pyroptosis, necroptosis, and autophagy.It is primarily driven by excessive intracellular iron and dysregulated lipid repair systems, leading to lipid peroxidation and morphological changes in mitochondria [8].During ferroptosis, the activity of glutathione peroxidase 4 (GPX4) is reduced, which appears to play a crucial role.Lower GPX4 expression increases susceptibility to ferroptosis, while higher expression is inhibiting [9,10] (Figure 1).

Regulating Ferroptosis
Ferroptosis is distinct from other forms of regulated cell death like apoptosis, pyroptosis, necroptosis, and autophagy.It is primarily driven by excessive intracellular iron and dysregulated lipid repair systems, leading to lipid peroxidation and morphological changes in mitochondria [8].During ferroptosis, the activity of glutathione peroxidase 4 (GPX4) is reduced, which appears to play a crucial role.Lower GPX4 expression increases susceptibility to ferroptosis, while higher expression is inhibiting [9,10] (Figure 1).Xc-functions as an amino acid antiporter, widely distributed within phospholipid bilayers.Comprising two subunits, solute carrier family 7 member 11 (SLC7A11) and solute carrier family 3 member 2 (SLC3A2), it forms a crucial part of the cellular antioxidant system.The exchange of cystine and glutamate occurs through System Xc-at a balanced ratio of 1:1, both entering and exiting the cell.Cystine, acquired through cellular uptake, undergoes reduction within cells and participates in glutathione (GSH) synthesis.GSH plays a role as an electron donor in reducing reactive oxygen species (ROS) and reactive nitrogen under the influence of GPXs, thereby forming oxidized GSSG out of two GSH molecules.Hampering the function of system Xc-influences the synthesis of GSH by impeding cystine absorption, leading to diminished GPX activity, reduced cellular antioxidant capacity, lipid ROS accumulation, and oxidative damage, culminating in ferroptosis.Further abbreviations are explained in the abbreviation table at the end of the review.
RAS-selective lethal 3 (RSL3) targets and suppresses GPX4 activity, leading to lipid ROS accumulation and ferroptosis induction [11].Erastin, initially identified for its selective toxicity in tumor cells, has gained attention as a ferroptosis inducer.Erastin triggers ferroptosis through various mechanisms involving system XC-, voltage-dependent anion channels, and p53 [12].General overview of the mechanisms of ferroptosis, irrespective of the cell type.System Xcfunctions as an amino acid antiporter, widely distributed within phospholipid bilayers.Comprising two subunits, solute carrier family 7 member 11 (SLC7A11) and solute carrier family 3 member 2 (SLC3A2), it forms a crucial part of the cellular antioxidant system.The exchange of cystine and glutamate occurs through System Xc-at a balanced ratio of 1:1, both entering and exiting the cell.Cystine, acquired through cellular uptake, undergoes reduction within cells and participates in glutathione (GSH) synthesis.GSH plays a role as an electron donor in reducing reactive oxygen species (ROS) and reactive nitrogen under the influence of GPXs, thereby forming oxidized GSSG out of two GSH molecules.Hampering the function of system Xc-influences the synthesis of GSH by impeding cystine absorption, leading to diminished GPX activity, reduced cellular antioxidant capacity, lipid ROS accumulation, and oxidative damage, culminating in ferroptosis.Further abbreviations are explained in the abbreviation table at the end of the review.RAS-selective lethal 3 (RSL3) targets and suppresses GPX4 activity, leading to lipid ROS accumulation and ferroptosis induction [11].Erastin, initially identified for its selective toxicity in tumor cells, has gained attention as a ferroptosis inducer.Erastin triggers ferroptosis through various mechanisms involving system XC-, voltage-dependent anion channels, and p53 [12].

Iron Metabolism
Iron accumulation is a key trigger of cytotoxic processes disrupting redox balance and leading to cell death [13].In the serum, transferrin serves as the primary iron transport protein [14].Iron-bound transferrin (Fe 3+ ) may bind to transferrin receptor 1 (TfR1), which is followed by uptake of complexed Fe 3+ into cells [3].Within endosomes, STEAP3 converts Fe 3+ to ferrous iron (Fe 2+ ), which is released into the labile iron pool (LIP) via divalent metal transporter 1 (DMT1, also known as SLC11A2).Most cytoplasmic Fe 2+ binds to ferritin light (FTL) and ferritin heavy chain (FTH), forming ferritin, mediated by chaperones like Poly-(rC)-binding protein 1 (PCBP1) and PCBP2 [3].Iron export is facilitated by FPN, the sole mammalian iron efflux pump [14].Ferroptosis-sensitive cells with Ras mutations show elevated TfR1 and reduced ferritin expression, suggesting that increased iron uptake and decreased iron storage contribute to iron overload during ferroptosis.Iron chelators like deferoxamine reduce iron overload and inhibit ferroptosis induced by molecules like erastin, while supplementing iron increases erastin-induced cell death.Knockdown of iron-responsive element-binding protein 2 (IREB2) increases expression of iron metabolism genes (e.g., FTH1 and FTL), inhibiting ferroptosis.Thus, as uptake and utilization systems of iron critically regulate ferroptosis, the availability of this metal is itself an essential component of the cellular response [14].

Lipid Peroxidation Pathways
Two critical metabolites for ferroptosis arise from lipid peroxidation, lipids and reactive oxygen species (ROS), which are primarily generated through the Fenton reaction [15].Free polyunsaturated fatty acids (PUFAs) may act as substrates in the synthetic lipid signaling pathway but must be esterified into membrane phospholipids and oxidized to transmit the ferroptotic signal.Arachidonic acid (AA)-containing phosphatidylethanolamine (PE) is a key phospholipid with the capacity to induce ferroptosis.Acyl-CoA synthetase longchain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) participate in PE biosynthesis and remodeling, activating PUFAs and influencing PUFA's transmembrane characteristics [16].In the non-enzymatic pathway, hydroxyl radicals (HO) strip lipids of hydrogen atoms (H•) to form lipid radicals, which create lipid peroxides [17].In the enzymatic pathway, iron-dependent lipoxygenases (LOXs) convert PUFAs into peroxides and derivatives like malonaldehyde (MDA) and 4-hydroxynonenal (4-HNE), both leading to ferroptosis [16].Lipid peroxidation can impair membrane-bound enzymes, altering plasma membrane fluidity and permeability [18].

Ferroptosis in Inflammatory Arthropathies
Joint inflammation (arthritis) is a hallmark of most rheumatic and musculoskeletal diseases (RMDs), which substantially affects the patient's body function and quality of life [19].There are more than 100 different, more specifically described clinical entities, all of which may go along with arthritis, but they importantly differ in their etiology, non-arthritic symptoms, severity, chronicity, and long-term outcome.Some types of arthritis have a known etiology like bacteria, crystals, or viruses, but the vast majority of arthritis is currently of unknown nature.Treatment of arthritis has been focused on certain immunological cell types and pro-inflammatory cytokines; however, there is an unmet need for other treatment avenues, since more than 50% of patients with immune-mediated arthritides do not respond satisfactorily to current treatment options [20].At present, researchers are increasingly recognizing potential connections between inflammatory arthritis and ferroptosis.This exploration holds promise in elucidating unknown components in the pathogenesis of inflammatory arthropathies, while unveiling prospective therapeutic targets for the future.
In this review, we will focus on RA, spondylarthritis (SpA), psoriatic arthritis (PsA), and crystal arthropathies, followed by a chapter about osteoarthritis (OA).
Table 1 summarizes current in vivo data from ferroptosis research in animal models with arthritis and from isolated and in vitro stimulated cell types from rats and mice.In addition, Table 2 summarizes ex vivo data observed in stimulated and non-stimulated human tissue from selected arthritis conditions.

Experiments in Animals
In Vivo In Vitro

Experiments in Humans
FLSs Chondrocytes

Detection of indicators for ferroptosis in arthritis
ACSF2, AURKA, EGFR, KLHL24 biomarkers ferroptosis in OA 30 samples of OA patients and 28 controls [60] Increased ROS levels RA-FLS, peripheral blood mononuclear cells from RA patients → ROS↑ in co-cultured RA-FLS and peripheral blood mononuclear cells [61] ROS detection in mild and severe OA regions of OA patients [62]
Researchers identified ferritin proteins (FTL, FTH) and iron transport proteins (like TfR and DMT1) in FLSs and macrophages from RA synovial tissue.They found that IL-6 and TNF-α enhance iron uptake by monocytes and FLSs from RA patients in vitro [68,73].The review will further explore the role of ferroptosis in FLSs within the context of RA.Neutrophils, essential cells of innate immunity, play a pivotal role in initiating and advancing arthritis.They are the most abundant immune cell in the synovial fluid of most types of arthritis but are only a minor population in synovial tissue.Previously viewed as a homogeneous cell population, we now recognize different neutrophil subsets with varying properties, even including immunosuppressive types [89,90].In arthritis, neutrophils experience increased cell survival and oxidative stress, thereby continuing to release neutrophil contents and form extracellular traps.These actions together with their interaction with other immune cells sustain inflammation and contribute to joint cartilage and bone degradation [91].Notably, despite their fundamental role, there is currently no published research on neutrophil ferroptosis in arthritis.

Macrophages and Dendritic Cells (DC)
There is evidence indicating that the accumulation of iron in inflammatory lesions worsens arthritis by triggering ferroptosis in macrophages.A positive correlation exists between elevated levels of iron in synovial fluid and the severity of RA, akin to the correlation observed between lipid hyperoxidation in specific macrophage populations and RA disease severity.Further investigation revealed that anti-inflammatory macrophages (M2) are highly susceptible to iron-induced ferroptosis, whereas pro-inflammatory macrophages (M1) are less affected.The ferroptosis inhibitor liproxstatin has been shown to mitigate the progression of K/BxN serum-transfer-induced arthritis in mice, accompanied by a shift towards M2 macrophages [50].

Ferroptosis in Adaptive Immune Cells 2.3.1. B Lymphocytes (B Cells)
B cells play a crucial role in linking the development of tertiary lymphoid tissue in inflamed synovium to the autoimmune process of RA, supported by the presence of germinal-center-like structures and the impact of B-cell-derived lymphotoxin-α on lymphoid architecture.CD4 T cell activation in the synovium depends on the presence of B cell follicles, with depletion of B cells hindering interferon-γ and IL-1 production, suggesting that other antigen-presenting cells cannot substitute for B cells in maintaining T cell activation [92].To date, there has been no research on B cell ferroptosis in arthritis.

T Lymphocytes (T Cells)
The synovium in the joints of patients with arthritis harbors various types of immune cells, with monocytes/macrophages and T cells being pivotal components.Monocytes/macrophages have the ability to attract and stimulate the differentiation of T cells into inflammatory phenotypes within the synovium.Similarly, distinct subtypes of T cells can attract monocytes/macrophages, fostering osteoclast differentiation and triggering the production of inflammatory cytokines [93].In RA synovial tissue, CD4 T cells were found to be lower compared to control synovial tissue, whereas the presence of CD8 T cells was increased [94].The only study found about T cell ferroptosis in arthritis showed that low doses of the neuroleptic haloperidol can suppress T cell ferroptosis in RA by decreasing the buildup of ferrous ions within lysosomes, resulting in a decreased production of intracellular ROS [95].

Rheumatoid Arthritis (RA)
RA is an immune-mediated inflammatory type of arthritis starting on some genetic background and possibly triggered by environmental factors.Clinically overt RA often occurs many years after the first detection of autoantibodies, indicating that currently unknown factors, other than autoantibodies and genes, are fundamental in this disease [96].

Iron Metabolism in RA
A study revealed disparities in iron metabolism between RA patients and healthy controls.In contrast to the less inflammatory type of arthritis in iron overload disease, disturbances in iron metabolism are not known as the primary event of immune-related arthritis.In contrast, probably secondary iron deficiency is prevalent (64%) among RA patients with elevated disease activity.RA patients exhibited decreased levels of hepcidin, transferrin saturation, and ferritin [97].It was also found that RA patients had notably elevated serum soluble transferrin receptor (sTfR) levels alongside significantly lower serum iron levels compared to the control group.sTfR demonstrated a significant positive correlation with parameters of inflammatory activity and autoimmune disease [98].One study examined the serum-derived proteomic alterations in patients classified as non-responders and responders 14 weeks after receiving a combination treatment of methotrexate + leflunomide + infliximab.Results revealed that serum transferrin levels were reduced at baseline in the non-responder group but elevated in the responder group.Further analysis suggests that serum transferrin plays a role in the hypoxia-inducible factor (HIF)-1 pathway and ferroptosis, potentially influencing the therapeutic outcome of this triple therapy [99].

Ferroptosis in RA
The impact of ferroptosis in RA is mainly dependent on the cell type undergoing ferroptosis.Here, we concentrate on the FLS, which are not only resident cells and candidates for the initial events of arthritis, but well-established key players in the RA joint and spreading of RA [100] (Figure 2).The central genes implicated in ferroptosis within RA synovium potentially include vascular endothelial growth factor A (VEGFA), prostaglandin endoperoxide synthase 2 (PTGS2), and JUN (transcription factor JUN), which are primarily associated with the FoxO signaling pathway [94].PTGS2, enolase 1 (ENO1), and granulin (GRN) were pinpointed and confirmed as plausible biomarkers associated with the regulation of ferroptosis.It was noticed that knocking down ENO1 resulted in heightened production of lipid ROS, greater buildup of intracellular ferrous ion, and increased cell mortality, as well as higher expression of aconitase 1 (ACO1).Research unveiled ENO1's elevated expression in RA synovium and suggested that ferroptosis might be governed by the ENO1-ACO1 axis [64].Another study screening the hub genes in RA found caspase-8 to be a significant biomarker for ferroptosis in RA as it was significantly increased in the ferroptosis phenotype group compared to the control group.Quercetin, a naturally occurring flavonoid, which exhibits strong antioxidant properties and has notable anti-inflammatory effects, can lower the caspase-8 levels, suggesting that it could be a potential treatment target for RA [101].

Ferroptosis Inducers in RA-FLS
Researchers found that imidazole ketone erastin (IKE) decreased FLS populations in CIA mice, leading to reduced concentrations of GPX4 in the remaining synovial tissue [25].On the other hand, sustained exposure to TNF-α, implicated as a key driver in RA pathogenesis, protected RA-FLSs from ferroptosis by enhancing cystine uptake and glutathione (GSH) synthesis.Conversely, erastin-induced ferroptosis worsened joint inflammation and disrupted gut microbiota and metabolites in CIA mice.Administering an antagonist 2 ′ (3 ′ )-O-(4-Benzoylbenzoyl) adenosine 5-triphosphate (BzATP) alleviated arthritic inflammation and abnormal intestinal microbiota caused by erastin [31].These results suggest that novel ferroptosis inducers used in combination with established TNF inhibitors could potentiate the therapy for RA.
Moreover, glycine was found to enhance ferroptosis in RA-FLSs by decreasing GPX4 levels and reducing FTH1 expression [68].
Conversely, small extracellular vesicle (sEV) production from RA-FLSs increased during ferroptosis induction due to local inflammation, led to elevated synovial VEGF expression, and enhanced angiogenesis.The release of sEV during ferroptosis may be linked to compensatory upregulation of the endosomal sorting complex required for transport (ESCRT-III), which aids in repairing cellular damage from ferroptosis stimulation.LPStreated FLSs showed increased ESCRT-III levels, and knockdown of the ESCRT-III subunit CHMP4A increased ROS levels and decreased GPX4 and SLC7A11 concentrations [70].This study highlights the importance of a repair system that has to be taken into account when inducing ferroptosis in FLS.
These diverse approaches of ferroptosis induction in RA-FLSs suggest that targeting synovial proliferation through ferroptosis may offer a new therapeutic avenue distinct from conventional immune-based treatments.

Ferroptosis Inhibitors in RA-FLS
Although ferroptosis inhibition in RA-FLSs is not a goal in view of their role in the disease, studies investigating ferroptosis inhibition in RA-FLSs have revealed interesting results, including the following.
These ferroptosis inhibitors might not be relevant for RA-FLSs but could possibly be used for other cell types such as chondrocytes in RA or OA.
Inducing ferroptosis in fibroblast-like synoviocytes (FLS) could shrink the synovial pannus, thus leading to the resolution of inflammation.However, the exact role of ferroptosis in RA is not fully established.Further detailed studies are needed to understand how the mechanism of ferroptosis can be utilized in the treatment of RA.

Spondylarthritis (SpA) and Other Arthritic Forms
Spondylarthritis (SpA) is another group of inflammatory diseases that primarily affect the joints in the spine.It has a similar morphology to synovitis but is linked to other genetic predispositions than RA.One type of SpA is psoriatic arthritis (PsA) [103,104].
PsA has more similarities with RA than other types, as it may also lead to bone erosion, but it has new bone formation in common with other types of SpA.Similarly to RA, the pathogenesis of PsA is largely influenced by proinflammatory cytokines, with key players including TNF-α and various interleukins, which have significant effects on joint structure [105].

Ferroptosis in Spondylarthritis
Only one published paper has focused on ankylosing spondylitis (AS) and ferroptosis.It demonstrated that patients with AS can be categorized into two distinct subtypes using ferroptosis-related genes (FRG)-based consensus clustering analysis.These subgroups exhibited clear differences in FRG expression patterns, as well as variations in immune cell compositions and enrichment of differentially expressed genes (DEGs) in pathways associated with mitochondria and ubiquitin [106].To analyze DEGs between these groups, the research team identified 12 hub genes and constructed a multifactorial regulatory network.Notably, the key nodes within this network were closely linked to redox homeostasis and the musculoskeletal system.The study indicates a potentially significant role of ferroptosis in the pathogenesis and molecular regulation of AS [107].However, further research in this area is needed to determine whether ferroptosis can be targeted in SpA.

Ferroptosis in Psoriatic Arthritis
Only one study has investigated ferroptosis in PsA, focusing on the association between ferroptosis regulators and key genes linked to PsA.The findings revealed a unique relationship between CDGSH iron sulfur domain 1 (CISD1), a ferroptosis regulator, and Ctype lectin domain family 2 member B (CLEC2B), a hub gene, in individuals with PsA [108].Further exploration is needed to determine if targeting this pathway could have therapeutic implications for PsA.

Ferroptosis in Crystal-Induced Arthritis
Crystal arthropathies in the case of gout and pseudogout are characterized by the accumulation of monosodium urate or calcium pyrophosphate dihydrate (CPPD) crystals in joints and surrounding tissues, leading to inflammation [109].
Research has validated the observed connection between higher levels of serum ferritin, iron, and elevated levels of serum urate.This provides evidence that elevated serum ferritin levels are positively linked to an increased risk of gout and more frequent gout flares.Mendelian randomization has shown evidence indicating a causal link between ferritin and iron in raising urate levels, but not the other way around.From a clinical perspective, the data imply that advising people with gout to avoid iron-rich foods could potentially help reduce the frequency of gout flares [110].According to a large-scale population study across China, there was a positive correlation between serum ferritin, TfR levels, and serum uric acid levels, as well as the likelihood of hyperuricemia [111].Iron's role in gout could be linked to xanthine oxidase (XO), which generates uric acid.Research indicates that iron might enhance the expression and function of XO [112].Additionally, certain cytokines like TNF-α and IL-6 could stimulate XO activation in bovine renal epithelial cells, leading to ROS production [113].It has been found that monosodium urate crystals can trigger various types of cell death, including ferroptosis, ultimately resulting in inflammatory cell death [114].
Currently, there is no study examining the impact of ferroptosis on crystal arthropathies, although evidence from epidemiological and mechanistic studies concerning the involvement of iron in gout suggest that ferroptosis could be an interesting pathway to investigate.

Ferroptosis in Osteoarthritis
OA is a form of arthritis that has been primarily linked to age-related wear and tear.In recent years, OA has been recognized as a multifaceted condition involving cartilage breakdown, bone remodeling, and joint inflammation.Risk factors include advancing age, female gender, obesity, prior joint injuries, anatomical abnormalities, and familial predisposition [115].The disruption of iron homeostasis is linked to cellular ferroptosis and degenerative diseases [72].Serum iron levels are higher in OA patients compared to controls, but transferrin expression and total iron binding capacity are diminished [39].Ferroptosis research in OA primarily targets chondrocytes, a cell type with an unequivocally central role in OA development (Figure 3).Very briefly, cartilage is composed of a dense network of collagen fibers embedded in an aggrecan gel, with a sparse population of cells known as chondrocytes.Chondrocytes play crucial roles in matrix production, repair, and remodeling.They respond to various signals, including growth factors, cytokines, and biomechanical forces to maintain cartilage health.Aging chondrocytes have an important impact on cartilage biology and pathology, affecting tissue function and resilience.Unlike other tissues, cartilage lacks mechanisms for cell replacement, making it vulnerable to irreversible damage from inherited factors and environmental stressors like trauma and obesity [36,116].
eling.They respond to various signals, including growth factors, cytokines, and biomechanical forces to maintain cartilage health.Aging chondrocytes have an important impact on cartilage biology and pathology, affecting tissue function and resilience.Unlike other tissues, cartilage lacks mechanisms for cell replacement, making it vulnerable to irreversible damage from inherited factors and environmental stressors like trauma and obesity [36,116].Researchers identified ferroptosis-related genes as diagnostic biomarkers and therapeutic targets for synovitis in OA, including EGFR.Inhibition of EGFR induced chondrocyte ferroptosis and matrix degradation, which was reversed by ferrostatin [60,79].NCOA4 expression is elevated in OA cartilage, aged mice, and mice with post-traumatic OA, driven by c-JUN N terminal kinase (JNK)-JUN signaling.Knocking down NCOA4 in IL-1β-treated chondrocytes reduced ferroptosis markers such as ACSL4 and p53, while increasing GPX4 and cell viability [72].
Another ferroptosis-related marker is sterol carrier protein 2 (SCP2), which is elevated in OA chondrocytes.SCP2 facilitated ferroptosis by transporting lipid peroxidation products to mitochondria, leading to cartilage degradation.Treating OA with a SCP2 inhibitor mitigated cartilage degradation and increased cell viability [29].Furthermore, long non-coding RNA maternally expressed 3 (lncRNA MEG3) was reduced in OA synovial fluid.Silencing lncRNA MEG3 decreased chondrocyte viability and increased ferroptosis markers.In contrast, increasing MEG3 reduced ferroptosis by modulating the miR-885-5p/SLC7A11 signaling pathway [60].Additionally, miRNA-1 expression was lower in OA cartilage compared to healthy cartilage, and its upregulation prevented ferroptosis in OA chondrocytes in a mouse model [53].Moreover, a study showed that stearoyl-CoA desaturase (SCD1) deficiency, a rate-limiting enzyme in the synthesis of unsaturated fatty acids, Researchers identified ferroptosis-related genes as diagnostic biomarkers and therapeutic targets for synovitis in OA, including EGFR.Inhibition of EGFR induced chondrocyte ferroptosis and matrix degradation, which was reversed by ferrostatin [60,79].NCOA4 expression is elevated in OA cartilage, aged mice, and mice with post-traumatic OA, driven by c-JUN N terminal kinase (JNK)-JUN signaling.Knocking down NCOA4 in IL-1β-treated chondrocytes reduced ferroptosis markers such as ACSL4 and p53, while increasing GPX4 and cell viability [72].
Another ferroptosis-related marker is sterol carrier protein 2 (SCP2), which is elevated in OA chondrocytes.SCP2 facilitated ferroptosis by transporting lipid peroxidation products to mitochondria, leading to cartilage degradation.Treating OA with a SCP2 inhibitor mitigated cartilage degradation and increased cell viability [29].Furthermore, long non-coding RNA maternally expressed 3 (lncRNA MEG3) was reduced in OA synovial fluid.Silencing lncRNA MEG3 decreased chondrocyte viability and increased ferroptosis markers.In contrast, increasing MEG3 reduced ferroptosis by modulating the miR-885-5p/SLC7A11 signaling pathway [60].Additionally, miRNA-1 expression was lower in OA cartilage compared to healthy cartilage, and its upregulation prevented ferroptosis in OA chondrocytes in a mouse model [53].Moreover, a study showed that stearoyl-CoA desaturase (SCD1) deficiency, a rate-limiting enzyme in the synthesis of unsaturated fatty acids, induced ferroptosis in chondrocytes.SCD1-ko mice developed early OA spontaneously, which further exacerbated in accelerated joint destruction after destabilization of the medial meniscus (DMM), an established model of mechanically induced OA [58].Conversely, in another set of experiments in the DMM mouse model, cyclin-dependent kinase inhibitor 1A (CDKN1A = p21) expression was increased in OA chondrocytes and in erastin-treated cells.However, p21 knockdown aggravated OA and exacerbated ferroptosis.Thus, p21 appears to exert a crucial anti-ferroptotic function in OA by modulating the stability of GPX4 [42].
Inflammatory and fibrocartilage chondrocytes in osteoarthritic cartilage showed activation of the ferroptosis pathway, along with increased iron-overload-related gene expression [117].This observation was confirmed by another study observing increased iron concentration, lipid peroxidation, and ferroptotic driver expression in damaged OA cartilage compared to intact areas.Single-cell RNA sequencing identified a distinct ferroptotic chondrocyte cluster, proposing transient receptor potential vanilloid 1 (TRPV1) as a potential anti-ferroptotic target in OA cartilage.Activating TRPV1 protected chondrocytes from ferroptosis and mitigated OA progression in a mouse model [59].Further, exosomal miR-19b-3p and miR-181b from OA-FLS, found to be increased in the cartilage of OA patients, promoted ferroptosis in mice chondrocytes, highlighting a potential link between synovium, cartilage, and ferroptosis in OA [27,71].
Interestingly, mechanical strain triggered ferroptosis in chondrocytes via piezo-type mechanosensitive ion channel component 1 (piezo1)-mediated calcium influx.Inhibiting piezo1 mitigated mechanical damage and ferroptosis [26].However, moderate mechanical stress slowed cartilage deterioration in a rat model and suppressed ferroptosis-related genes by triggering the Nrf2 antioxidant system [80].Thus, the amount of mechanical stress might play a role in the induction of ferroptosis in cartilage.
Similarly, increased expression of staphylococcal nuclease domain-containing 1 (SND1) in IL-1β-stimulated chondrocytes led to GPX4 degradation.Silencing SND1 in these chondrocytes and in a DMM rat model reduced TNF-α levels and iron concentrations and increased GPX4 expression [28].
These studies highlight IL-1β as a possible trigger for ferroptosis induction.

Ferroptosis Inhibition in Unstimulated Chondrocytes
In human cartilage explants, cellular markers of ferroptosis were positively correlated with the severity of cartilage damage and MMP-13 expression.When chondrocytes from mild OA cartilage were treated with Fer-1, cells showed improved activity and mitochondrial function and lower MMP13 expression, increased concentrations of GPX4 as well as SLC7A11, but diminished ACSL4 and p53 levels, whereas cells from moderate to severe OA could not be rescued in their function by ferroptosis inhibition [44].This is an interesting finding suggesting ferroptosis might play a role in the beginning of the disease.
There are several studies exploring the effect of plant extracts on ferroptosis inhibition in OA chondrocytes.In a mouse model, curcumin, an extract derived from turmeric rhizomes, prevented cartilage breakdown induced by erastin.Silencing Nrf2 reversed the beneficial effects of curcumin, suggesting a role in enhancing chondrocyte resistance via inhibition of ferroptosis [40].Similarly, acetyl zingerone (AZ), a curcumin derivative, increased chondrocyte viability and proliferation in another DMM model, apparently suppressing ferroptosis by promoting GPX4 expression and Nrf2/HO-1 activation [41].Likewise, theaflavin-3,3 ′ -digallate, extracted from black tea, protected rat and human chondrocytes against erastin-induced ferroptosis via Nrf2/GPX4 pathway activation [32].
Concerning drugs, calcipotriol, a synthetical vitamin D analogue, reduced cartilage damage by inhibiting GPX4-mediated ferroptosis and suppressing TGF-β1 and lipid peroxidation of chondrocytes in a DMM mouse model [38].The antidiabetic drug metformin mitigated OA-associated histopathological changes and abnormal angiogenesis in subchondral bone in a DMM model [52], while pioglitazone, a peroxisome-proliferator-activated receptor γ (PPARγ) agonist, increased GPX4 via PTEN-induced kinase 1 (Pink1)/Parkindependent mitophagy to protect RSL3-treated rat chondrocytes from ferroptosis [55].Conversely, paxlovid, a drug that inhibits the synthesis of virus-related proteins and replication of viral RNA, induced ferroptosis in chondrocytes and accelerated their senescence and degeneration in a mouse model of OA [121].
In summary, inhibiting ferroptosis in OA chondrocytes appears to be beneficial for the outcome of several models of OA.However, the pharmacological compounds appear to be very heterogeneous, and in particular, the potentially active compounds in the phytopharmacological intervention are not well described.Thus, further investigation is needed to determine if any of these approaches may be effective and safe in humans as well.

Interpretation of the Data
To summarize the results of our review, we found accumulating evidence that ferroptosis might be a key event in the initiation of cartilage pathologies.In ex vivo studies on human cartilage explants, in metabolically or mechanically induced models of OA in rodents, we found consistent evidence that ferroptosis might be a relevant component in the breakdown of cartilage.Thus, it appears to be desirable to pharmacologically inhibit ferroptosis in early OA.However, OA-related interventions were heterogeneous, the sample size of the studies is usually small, and data are not yet clear enough to propagate a certain intervention.This is particularly true for most of the smaller studies with plant-derived compounds.
However, while inhibition of ferroptosis appears to be desirable in resident cartilage cells to prevent OA, the ferroptosis inducer erastin was able to improve inflammatory arthritis severity in CIA mice [31,40,50].It appears that in this model of arthritis, inhibition of ferroptosis in FLS, another type of resident joint cells (like chondrocytes), might have detrimental effects.
Thus, it is currently not fully clear whether ferroptosis might be a physiological and beneficial process for tissue integrity, or a critical step in arthritis pathogenesis worth being treated.From the current literature, the impact of ferroptosis appears to strongly rely on mechanical or immunological stimuli and cell type.The current literature indicates that inhibiting ferroptosis in anti-inflammatory macrophages, chondrocytes, and T-cells leads to reduced inflammation in the joints of rheumatoid arthritis (RA) and osteoarthritis (OA) and related models.Conversely, inducing ferroptosis in RA fibroblast-like synoviocytes (FLS) appears to have a positive effect, alleviating inflammation.

Conclusions and Future Perspectives
Although the current state of research suggests an important role for ferroptosis in arthritis, the origins of the literature are currently not globally distributed but restricted mostly to Asian countries.Additionally, confirmation studies are often missing.However, this scarcity of research may be due to the relatively young nature of this topic.We identified a relevant gap in the research on ferroptosis in some prevalent and well-defined human diseases with known pathogenesis, such as crystal-induced arthritis.Moreover, given the major role of FLSs despite only being established in the pathogenesis of RA, it is tempting to speculate that the effect of inhibiting ferroptosis on the severity of synovitis in SpA and PsA might be fundamentally different from RA.
A limitation of our review may be that the search terms were restricted to title or abstract words, possibly leaving out some studies.Writing this review, we realized that ferroptosis induction or inhibition on a whole organism is restricted to rodents, thus leaving us with only cell-type-specific studies for human data.Since the still-preliminary results on the effect of ferroptosis in the development of RA and OA appear to demonstrate opposite consequences in disease-relevant cell types, we assume that ferroptosis does not explain the development of arthritis in general but can account for certain aspects of its pathogenesis.
In summary, the involvement of ferroptosis in different types of arthritis is cell-typespecific and requires careful evaluation for new treatment approaches.Further in vivo studies are needed to understand how ferroptosis can be utilized in the treatment of RA and OA.Given the consistent data on chondrocyte biology and the limited treatment options, especially in OA, targeting ferroptosis in chondrocytes might be a promising strategy to reduce the global burden of this prevalent disease in the aging population.

Conflicts of Interest:
The authors declare the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Figure 1 .
Figure 1.General overview of the mechanisms of ferroptosis, irrespective of the cell type.System Xc-functions as an amino acid antiporter, widely distributed within phospholipid bilayers.Comprising two subunits, solute carrier family 7 member 11 (SLC7A11) and solute carrier family 3 member 2 (SLC3A2), it forms a crucial part of the cellular antioxidant system.The exchange of cystine and glutamate occurs through System Xc-at a balanced ratio of 1:1, both entering and exiting the cell.Cystine, acquired through cellular uptake, undergoes reduction within cells and participates in glutathione (GSH) synthesis.GSH plays a role as an electron donor in reducing reactive oxygen species (ROS) and reactive nitrogen under the influence of GPXs, thereby forming oxidized GSSG out of two GSH molecules.Hampering the function of system Xc-influences the synthesis of GSH by impeding cystine absorption, leading to diminished GPX activity, reduced cellular antioxidant capacity, lipid ROS accumulation, and oxidative damage, culminating in ferroptosis.Further abbreviations are explained in the abbreviation table at the end of the review.

Figure 1 .
Figure 1.General overview of the mechanisms of ferroptosis, irrespective of the cell type.System Xcfunctions as an amino acid antiporter, widely distributed within phospholipid bilayers.Comprising two subunits, solute carrier family 7 member 11 (SLC7A11) and solute carrier family 3 member 2 (SLC3A2), it forms a crucial part of the cellular antioxidant system.The exchange of cystine and glutamate occurs through System Xc-at a balanced ratio of 1:1, both entering and exiting the cell.Cystine, acquired through cellular uptake, undergoes reduction within cells and participates in glutathione (GSH) synthesis.GSH plays a role as an electron donor in reducing reactive oxygen species (ROS) and reactive nitrogen under the influence of GPXs, thereby forming oxidized GSSG out of two GSH molecules.Hampering the function of system Xc-influences the synthesis of GSH by impeding cystine absorption, leading to diminished GPX activity, reduced cellular antioxidant capacity, lipid ROS accumulation, and oxidative damage, culminating in ferroptosis.Further abbreviations are explained in the abbreviation table at the end of the review.

28 Figure 2 .
Figure 2. Experiments on ferroptosis in rheumatoid arthritis.Animal experiments are marked in blue.Experiments on isolated human fibroblast-like synoviocytes (FLS) are marked in pink.Abbreviations are explained in the glossary.2.4.3.Ferroptosis Inducers in RA-FLSResearchers found that imidazole ketone erastin (IKE) decreased FLS populations in CIA mice, leading to reduced concentrations of GPX4 in the remaining synovial tissue[25].On the other hand, sustained exposure to TNF-α, implicated as a key driver in RA pathogenesis, protected RA-FLSs from ferroptosis by enhancing cystine uptake and glu-

Figure 2 .
Figure 2. Experiments on ferroptosis in rheumatoid arthritis.Animal experiments are marked in blue.Experiments on isolated human fibroblast-like synoviocytes (FLS) are marked in pink.Abbreviations are explained in the glossary.

Figure 3 .
Figure 3. Experiments on ferroptosis in osteoarthritis.Animal experiments for inhibition or induction of ferroptosis in vivo or in vitro are marked in purple.Experiments on human chondrocytes in vitro are marked in green.Abbreviations are explained in the glossary.

Figure 3 .
Figure 3. Experiments on ferroptosis in osteoarthritis.Animal experiments for inhibition or induction of ferroptosis in vivo or in vitro are marked in purple.Experiments on human chondrocytes in vitro are marked in green.Abbreviations are explained in the glossary.

Author
Contributions: S.B., J.A. and B.M. wrote the manuscript.S.B. generated the figures, the tables, and the glossary.All authors have read and agreed to the published version of the manuscript.Funding: This research received no external funding.

Table 1 .
Effects of induction and/or inhibition of ferroptosis in animal models with arthritis.In vitro studies concentrate on rodent chondrocytes and synovial fibroblasts.They demonstrate that induction of ferroptosis in synovial fibroblasts by different agents decreases the viability of synovial fibroblast and GPX4 expression.On the other hand, inhibition of ferroptosis in chondrocytes increases cell viability and decreases lipid peroxidation and cartilage degradation.↑: upregulated; ↓: downregulated; →: results in.
Innate Immune Cells 2.2.1.Polymorphonuclear Neutrophils (PMN) Is a member of Gli-Kruppel zinc finger proteins, plays a role in inhibiting and activating gene promotors